MX2007001133A - Use of vitamin d receptor activators or vitamin d analogs to treat cardiovascular disease. - Google Patents

Abstract

Disclosed are pharmaceutical compositions containing Vitamin D receptor activators or Vitamin D analogs to treat, prevent or inhibit vascular disease among other conditions. The pharmaceutical compositions may also include ACE inhibitors or other agents. Also disclosed are methods of reducing PAI-1 expression by administering effective amounts of Vitamin D receptor activators or Vitamin D analogs to a mammal in need thereof. Additionally disclosed are methods of preventing, inhibiting or treating thrombosis in a mammal in need of such prevention, inhibition or treatment comprising administering effective amounts of Vitamin D receptor activators or Vitamin D analogs to the mammal.

Description

USE OF VITAMIN D RECEIVING ACTIVATORS OR VITAMIN D ANALOGS TO TREAT DISEASES CARDIOVASCULAR Field of the Invention The present invention relates to the use of a vitamin D receptor activator (VDRA), preferably paricalcitol, or a vitamin D analog, to treat and prevent cardiovascular diseases, including cerebrovascular and peripheral vascular diseases, especially malfunctions of the heart, cardiomyopathy, atherosclerosis, myocardial infarction, and cerebrovascular accidents.

Background of the Invention Complications of cardiovascular diseases (CVD) due to atherosclerosis and cardiomyopathy are the most common cause of death in Western societies. Hypertension and hyperlipidemia in particular are major cardiac risk factors. Certain medications that treat hypertension (for example, angiotensin-converting enzyme inhibitors (ACEIs)) and abnormal lipid levels have been shown to reduce cardiovascular mortality significantly in high-risk populations, such as hemodialysis patients. However, several factors, including adverse side effects, limit the usefulness of existing drugs to prevent the progression of cardiovascular disease or otherwise render these drugs inadequate for the treatment of CVD, particularly critical for high-risk populations. The biological effects of VDRAs are mediated through the vitamin D receptor (VDR), a member of the nuclear hormone receptor superfamily. A mechanism through which VDR is thought to mediate biological effects is through the activation of a transcription factor that binds to specific elements of DNA sequence in genes sensitive to vitamin D and ultimately has an influence on the speed of transcription mediated by RNA polymerase II. VDRs are present in most types of human cells, especially in the cardiovascular system and immune system. Several lines of evidence support the idea that vitamin D plays an important role in the regulation of cardiovascular physiology as described in Figure 1. Vitamin D has the potential to prevent arteriosclerosis and vascular calcification through its effects on the immune system to down-regulate inflammatory trajectories and to restore normal expression in vascular calcification meters. Vitamin D also affects the proliferation of cells. Low levels of vitamin D were associated with congestive heart failure. Vitamin D has direct effects to antagonize cardiomyocyte hypertrophy induced by endothelin-1. Finally, VDRAs down regulate RAAS by inhibiting renin synthesis. Thus, treatment with VDRAs / vitamin D analogues can prevent or treat cardiovascular disease by affecting one or all of the trajectories in Figure 1. However, in vitro and animal data have suggested that VDRAs and / or vitamin analogs D can damage the heart in uraemic patients, for example, causing vascular calcification, myocardial infarction, heart failure, cardiomyopathy and cerebrovascular accidents. Therefore, the medical community does not support the use of these compounds as a therapy for cardiovascular disease and recommends limiting their use.

Brief Description of the Invention The present invention is directed to methods for preventing, treating and delaying the progression of vascular diseases, including cardiovascular, cerebrovascular and peripheral vascular diseases, especially heart failure, cardiomyopathy, arteriosclerosis, myocardial infarction, and cerebrovascular accidents, and pharmaceutical compositions useful for this. According to one embodiment, the present invention relates to VDRAs or vitamin D analogues, (hereinafter referred to as "VDRA / vitamin D analogue"), containing compositions for preventing, treating and delaying the progression of vascular diseases. According to some aspects of the present invention, vitamin D receptor activating compounds (VDRAs) can be used. VDRAs include paricalcitol, calcitriol, 22-oxa-1-alpha, 25-dihydroxyvitamin D2, MC-903 (calcipotriol), 16-en-23-in-1alpha, 25-dihydroxyvitamin D3, and 24-difluoro-26,27-dimethyl-16-en-1alpha, 25-dihydroxyvitamin D3 (described in more detail by DeLuca, et al. ., in PNAS, 2004, vol.101, No.18, p.6900-6904, incorporated herein by reference), the compounds listed in Table 1 of Physiol. Rev. October 1998, Vol. 78, No. 4, p 193-1231, incorporated herein by reference, and the so-called Gemini compounds (described in more detail by Maehr, et al., In J. Steroid Biochem. Mole. 89-90, 2004, 35-38, incorporated herein by reference), EB-1089 (a pharmaceutical compound of LEO), and ED-71 (a Roche compound). Paricalcitol is especially preferred since it is a selective VDRA. Paricalcitol is commercially available from Abbott Laboratories (North Chicago, IL, under the trade name of ZEMPLAR). In accordance with other aspects of the present invention, the vitamin D analog may be doxercalciferol or alfacalcidol. According to some embodiments, the especially preferred compositions of the present invention also include one or more of the following agents: angiotensin-converting enzyme inhibitor (ACEL) or an angiotensin receptor blocker 11-1 (AT1) or a blocker of aldosterone (ARB). The compositions according to the present invention may also include other agents used to treat or prevent cardiovascular diseases, such as beta blockers, calcium channel blockers, antilipemic agents, antihypertensive agents and anti-inflammatory agents, including aspirin. According to some aspects of the invention, pharmaceutical compositions can be administered through a sustained (or continuous) delivery system. The present invention also contemplates other modes of administration, including, but not limited to, oral, injectable and transdermal. The present invention also includes a method of treating, inhibiting or preventing thrombosis in a mammal in need of such treatment, inhibition or prevention, comprising the step of administering to the mammal an effective amount of a vitamin D receptor activator or an analogue. of vitamin D. The activator of vitamin D receptor can be, for example, paricalcitol or calcitriol, and the vitamin D analog can be, for example, doxercalciferol or alfacalcidol. All patents and publications referred to herein are incorporated herein by reference in their entirety.

Brief Description of the Drawings Figure 1 schematically depicts the role of vitamin D in the de-regulation of various inflammatory factors associated with atherosclerosis and its association with cardiomyocyte remodeling. Figure 2 represents bar graphs comparing average hospitalizations per year and days in hospital per year for paricalcitol, calcitriol and without therapy with D.

Figure 3 presents graphs in the form of bars comparing the results of regression analysis showing treatment with paricalcitol associated with very few hospitalizations and days in the hospital compared to no vitamin D. Figure 4 shows a Northern graph, which Evidence that treatment with paricalcitol of As4.1-hVDR cells depends on dose dependently inhibits the expression of renin mRNA. Figure 5 illustrates the results of a renin-luciferase promoter assay used to examine the activity of paricalcitol to suppress transcription of the renin gene. Figure 6 illustrates the effect of paricalcitol and calcitriol on PAI-1 in a primary culture of human coronary artery smooth muscle cells. Figure 7 illustrates the effect of vitamin D analogues on the expression of the NPR-A gene promoter. VD3 represents 1.25 dihydroxyvitamin D (all results are normalized for the expression of CMV renilla luciferase co-transfected). Figure 8 shows the effect of vitamin D analogues on accumulation of cyclic GMP stimulated by ANP, where the generation of ANP-dependent cGMP was used as a substitute for ANP activity. Figure 9 shows the effect of vitamin D analogues on a mutant NPR-A gene promoter (VDRE-deleted) in neonatal rat aortic smooth muscle cells; the results are normalized for Renilla luciferase expression. The results suggest that all these tested compounds induce ANP through! Vitamin D response element. Figure 10 shows the effect of vitamin D analogues at baseline against the promoter activity of the endothelin-stimulated hBNP gene (10"7 M) using transfected cardiac myocytes that were cultured in a serum-free medium. Figure 11 shows the effect of vitamin D analogues on basal and promoter activity of the stimulated hBNP gene (10"7 M) by endothelin using transfected cardiac myocytes cultured in 0.2% fetal bovine serum. All cells were co-transfected with expression vectors directing the expression of hVDR and hRXR. Figure 12 shows the effect of vitamin D analogues on basal and Cdk2 activity stimulated by endothelin (10"7 M) in neonatal rat aortic smooth muscle cells.

Description of the Modalities of the Invention The present invention is generally directed to compositions containing VDRA / vitamin D analog, to treat or prevent cardiovascular diseases (CVD), including cardiomyopathy, coronary arterial, cerebrovascular and peripheral vascular diseases. The present invention also relates to methods for treating CVD by administering to a patient a pharmaceutical composition, which may be a sustained release formulation, containing a therapeutically effective amount of a VDRA / vitamin D analog. Treatment of patients with CVD through the administration of a therapeutically effective amount of a composition containing VDRA / vitamin D analogue is expected to be advantageous for an effective reduction of renin expression, reduced inflammation and improved cardiac function directly through the therapeutic action of VDRA / Vitamin D analogue in cardiac tissue. In contrast, conventional treatments that rely on the administration of an ACER (ie, without a VDRA / vitamin D analogue) for example, only reduce angiotensin (II), but do not reduce renin levels or act on receptors of vitamin D in the heart, vasculature and the same immune system. The administration of ACEL may not be an attractive treatment in the long term due to adverse consequences. In accordance with some aspects of the present invention, the compositions of the invention contain a VDRA / vitamin D analogue and at least one of the following agents: an ACE inhibitor, an angiotensin (II) receptor blocker (ARB) ) and an aldosterone blocker in therapeutically effective amounts to inhibit renin production or to inhibit the activation of the renin-angiotensin-aldosterone system. Preferred compositions contain paricalcitol with at least one of these other agents. Such combinations may prevent the escape of ACE inhibition and escape of aldosterone with subsequent increase in generation of angiotensin (II) and aldosterone. Suitable ACE inhibitors, ARB blockers and aldosterone are commercially available. ACE inhibitors include, but are not limited to: captopril (commercially available under the tradename CAPOTEN from Mylan), enalapril (commercially available under the tradename VASOTEC from Merck), fosinapril (commercially available under the trade name MONOPRIL) from Bristol Myers Squibb), benzapril (commercially available under the trade name LOTENSIN from Novartis Pharmaceuticals), moexipril (commercially available under the trade name UNIVASC from Schwarz Pharma), perindopril (commercially available under the trade name ACEON from Solvay), quinapril (commercially available under the tradename ACCUPRIL from Parke-Davis), ramipril (commercially available under the trade name ALTACE from Monarch), trandolapril (commercially available under the tradename MAVIK from Abbott Laboratories of North Chicago, IL), lisinopril (commercially available under the trade names of PRINIVIL de and ZESTRIL de Astr to Zeneca). Suitable angiotensin receptor blocking agents, include, but are not limited to: losartan (commercially available as Merck's COZAAR), irbesartan (commercially available as AVAPRO from Bristol Myers Squibb and Sanofi), candesartan (commercially available as ATACAND from Astra Zeneca), eprosartan (commercially available as TEVETEN from Biovail Corporation of Canada), telmisartan (commercially available as MICARDIS from Boehringer Ingelheim) and valsartan (commercially available as DIOVAN from Novartis). Suitable aldosterone blockers include, but are not limited to: eplerenone (commercially available under the brand name of Pharmacia INSPRA), spironolactone (commercially available under the trade names of Aldactone, Adultmin, Aldopur, Aldospirone, Almatol, Berlactone, Diatensec, Diram, Esekon, Hypazon, Idrolattone, Merabis, Novospiroton, Osiren, Osyrol, Pyrolacton, Resacton, Sincomen, Spiractin, Spiroctan, Spirolacton, Spirolang, Spironex, Spirotone, Tevaspirone, Verospíron , Xenalon Lactabs, Youlactone). Additional components, e.g., physiologically acceptable carriers, solvents, binders, antioxidants, dyes, and substrates, may be used as needed or desired. The preferred treatment or preventive regimens for patients with CVD in accordance with the present invention could administer therapeutically effective compositions containing VDRA / vitamin D analogue according to the invention for a period sufficient to effect a sustained sustained delivery. As used herein, a "therapeutically effective dose" is a dose wherein in susceptible subjects it is sufficient to prevent progression or cause regression of CVD or that is capable of relieving symptoms caused by CVD. An illustrative dosage regimen could provide the equivalent of approximately 0.5 micrograms of calcitriol per day or at least about 1 microgram of calcitriol by injection three times per week. For paricalcitol, a suitable dosage regimen could provide the equivalent of about 2 micrograms of paricalcitol daily or at least about 4 micrograms of paricalcitol three times a week administered as a bolus. Suitable dosage regimens for VDRA / vitamin D analogues, eg, doxercalciferol, can be determined directly by those skilled in the art based on the therapeutic efficacy of the VDRA / vitamin D analogue to be administered. Since inhibitors of ACEl, ARB and aldosterone have different efficiencies and affect the body through different trajectories than vitamin D does, the compositions according to the present invention may incorporate an inhibitor of ACEL, ARB or aldosterone to be administered according to conventional dosing regimens, which are well known and readily available to those skilled in the art. The invention contemplates sustained or sustained drug delivery forms, containing the VDRA / vitamin D analogue selected, and a blocker of ACEL and / or ARB and / or aldosterone. Suitable delivery forms include, but are not limited to, tablets or capsules for oral administration, injections, transdermal patches for topical administration (for example the drug to be administered is mixed with a polymer matrix adhered to or absorbed in a support or substrate. back-up, eg, ethylcellulose), reservoirs (e.g., injectable microspheres containing the desired compounds and actives) and implants. The techniques for making these drug delivery forms are well known to those skilled in the art. In addition, it should also be noted that patients with CKD who undergo hemodialysis usually require the formation of an arteriovenous fistula (A-V) for hemodialysis (HD). Autologous A-V fistula has been proven as the most durable access for HD. The main failure of vascular access mainly refers to thrombosis. The pathophysiology underlying stenosis formation is blood flow turbulence that activates platelets and endothelial cells. The final activation that causes thrombosis is a critical reduction in the flow of blood from the fistula. In this context, a particular role for the platelet-derived growth factor (PDGF) has been postulated. Based on the data presented in Example 5 below, it can be concluded that it can be concluded that there is a statistically significant association with Zemplar therapy and fewer vascular access changes. In this way, Zemplar can have a beneficial effect through its action on endothelial cells, platelets and PDGF that are responsible for thrombosis.

Future studies should clarify the mechanism of the proposed effect, understood to extend beyond AV fistulas to grafts, dose-time dependence and association with the CaxP product. The present invention is further illustrated through the use of the following non-limiting examples: Example 1 Reduced morbidity and mortality associated with vitamin D therapy The main cause of mortality and morbidity in patients receiving chronic hemodialysis in relation to cardiovascular disease. The prevalence of CVD can be found in at least 75% of patients who initiate hemodialysis therapy. An observational initiation study examining hemodialysis patients who initiated vitamin D therapy with paricalcitol experienced fewer hospitalizations in relation to cardiovascular events and there were no infectious inflammations, with patients treated with calcitriol (Paricalcitol-treated patients experience improved hospitalization outcomes compared with calcitriol -treated patients in real-world clinical settings, DG Dobrez, et al., Nephrol Dialysis Transplant 2004 19: 1174). This study was expanded to include patients who did not receive any vitamin D activating treatment ["improved hospitalization outcomes in hemodialysis patients treated with paricalcitol." J. Melnick, et al., Abstract book from World Conqress of Nephroloqy, June 8-12, 2003, Berlin. Page 148] revealed that treatment with paricalcitol was associated with improved hospitalizations in patients with hemodialysis (HD) who were treated with paricalcitol or with calcitriol compared to patients who did not receive any treatment with vitamin D. As shown in Figure 2, the evaluation of hospitalization endpoints reveals the average number of hospitalizations in a year for patients who received VDRA (either paricalcitol ("Par") or calcitriol ("Cal")) was lower than for patients who did not receive any vitamin D ("Without vitamin D"). Notably, hospitalizations were lower for patients treated with paricalcitol (1.5) than for those treated with calcitriol (2.2). In addition, the average number of days in the hospital was lower for patients who received VDRA (especially paricalcitol) compared to patients who did not receive any vitamin D (2.6). The number of days in the hospital was again lower for paricalcitol (5.2) compared with calcitriol (10.6) and without vitamin D (14.7). Figure 3 presents multivariable results for hospitalizations and days in the hospital per year. The regression analysis of these data revealed that those receiving calcitriol were associated with 7.7 fewer days of hospitalization compared with the group that did not receive vitamin D, although there was no statistical difference in the number of hospitalizations. However, treatment with paricalcitol was associated with 1.2 fewer hospitalizations and 17.5 fewer days in the hospital compared to the group that did not take vitamin D.

Example 2 Activity of Paricalcitol to Suppress Renin Expression Recently, 1,2-dihydroxyvitamin D has been found to function as a negative regulator of renin biosynthesis in in vitro and in vivo studies. Calcitriol is able to inhibit renin gene expression, which provides a molecular basis for exploring the use of vitamin D and vitamin D analogues as a new renin inhibitor to regulate the renin-angiotensin-aldosterone system (RAAS) . When using an in vitro cell culture system, the activity of paricalcitol was examined to suppress renin gene expression using previously published techniques (1,25-Dihydroxyvitamin D3 is a negative endocrine regulator of the renin-angiotensin system, J. Clin. Invest., July 2002). As shown in Figure 4, through Northern stain analysis, paricalcitol treatment of As4.1-hVDR cells dependently on the dose inhibits the expression of renin mRNA. Actually, its renin inhibitory activity seems a more potent point than calcitriol (Figures 4A and B). This inhibitory effect is confirmed by promoter-luciferase report assays, which examine the activity of paricalcitol to suppress renin gene transcription. In these assays, paricalcitol appears to be at least as potent as calcitriol to suppress the activity of the renin gene promoter (Figure 6). These data support the usefulness of a VDRA / vitamin D analogue to regulate the renin-angiotensin-aldosterone system and its criticism in the development of CVD and delay in the progression of cardiovascular diseases.

Example 3 Effect of VDR Activators on PAI-1 The effect of paricalcitol and calcitriol on PA1-1 was investigated in a primary culture of human coronary artery smooth muscle cells (See Figure 6). EI PAI-1 (plasminogen activator inhibitor type 1) is one of the risk markers for coronary heart disease, and is improved in atherosclerotic plague and co-localized with macrophages. Human coronary artery smooth muscle cells were incubated with paricalcitol or calcitriol at the indicated concentration for 24 hours at 37 ° C. The samples were solubilized in SDS-PAGE sample pH buffer, and the protein content in each sample was determined through the Bio-Rad dye binding protein assay. The cells were resolved through SDS-PAGE using a 4-12% gel, and the proteins were electrophoretically transferred to a PVDF membrane for Western staining. The membrane was shaken for 1 hour at 25 ° C with dry milk without 5% fat in PBS-T and then incubated with a mouse anti-PAl-1 monoclonal antibody in PBS-T overnight at 4 ° C. . The membrane was washed with PBS-T and incubated with an anti-mouse antibody labeled with horseradish peroxidase for 1 hour at 25 ° C. The membrane was then incubated with a detection reagent (SuperSignal WestPico). The specific bands were visualized by exposing the paper to Kodak BioMax films. Figure 6 shows the results of Western staining using an anti-PAI-1 antibody. In these studies two observations can be observed: (1) 100% growth inhibition was never achieved at a concentration of 1 μM of any test compound. Focal microscope studies confirm that although these drugs are potent for inducing VDR translocation from the cytoplasm to the nucleus, not all cells respond to VDRAs even after a 2-hour exposure, which may explain the inhibition of < 100% (2) Although it is known that paricalcitol is less potent than calcitriol in clinical studies, it exhibits a potency similar to calcltriol in this assay. When verifying the effect of drugs on 24 (OH) ase expression, paricalcitol was found to be less potent than calcitriol in stimulating the expression of 24 (OH) ase, which partially explains the superior potency of paricalcitol in this assay. . These results show that paricalcitol and calcitriol are equally potent in reducing the level of PAI in smooth muscle cells of the human coronary artery. Paricalcitol is usually dosed approximately 4 times more than calcitriol in the clinical situation, which can be translated to a power of 4 times more to regulate the function of smooth muscle cells.

EXAMPLE 4 Effect of Paricalcitol on In Vitro Models Using Myocardial or Vascular Smooth Muscle Cells in Culture Experimentally induced vitamin D deficiency was associated with cardiac hypertrophy and hypertension in otherwise normal adult Sprague-Dawley rats (Weishaar et al. , Am, J. Phvsiol, 1990 Jan; 258 (1 Pt 1): E134-42). Cardiac hypertrophy can also be seen in the VDR - / - mouse (Li et al., J. Clin, Invest, 2002 Jul; 110 (2): 229-38), although this occurs in the determination of an elevation of 10. -15 mm Hg in the systolic blood pressure implying that the hypertrophy can, at least in part, reflect an increased ventricular overload. Vitamin D has been shown to inhibit endothelin-induced hypertrophy (ET) of neonatal rat cardiac myocytes in culture (Wu et al., J. Clin.Invest 1996 Apr 1; 97 (7): 1577-88 and Li et al. , J. Biol. Chem. 1994 Feb 18; 269 (7): 4934-9). This is associated with a reduction in the expression of ANP, BNP and skeletal actin genes and suppression of the human ANP and BNP gene promoters (Wu et al., Am. J. Phvsiol., 1995 Jun; 268 (6 Pt 1). : E1108-13 In this study, an examination was made of whether paricalcitol has similar effects (against the native hormone) in several in vitro models using myocardial or vascular smooth muscle cells, in culture.

Effect of VDRA / vitamin D analogues on the activity of the promoter of the NPR-A gene. Neonatal RASM cells were transfected with 1575 NPR-A LUC (0.5 μg) through electroporation. The cells were co-transfected with a report of constitutively active CMV-Renilla luciferase (0.25 μg) to control the differences in transfection efficiency. After 24 hours of transfection, the cells were treated with vitamin D analogues, or with vehicle, as indicated. The incubation was continued for 48 hours at that point the cells were harvested, the lysates were generated and measurements of luciferase (firefly and Renilla) were made.

Effect of VDRA / vitamin D analogues on NPR-A activity Cells were pre-incubated for 48 hours in 1,25 dihydroxyvitamin D (VD), paricalcitol, HECTEROL® (calcitriol) or the activated form of HECTEROL (calcitriol). At that point, the medium was changed, the non-selective phosphodiesterase inhibitor IBMX (10"4 M) was added, and the incubation was continued for 10 minutes at 37 ° C. Then ANP (10" 7 M) was added to each Culture and incubation was extended for 10 more minutes. Then, the medium was aspirated, the cells were smoothed with TCA and soluble extracts subjected to ether extraction, neutralization and radioimmunoa for cGMP levels. All cGMP levels presented here are normalized per μg of soluble protein present in the extract. The results are shown in Figures 7, 8 and 9.

Effect of vitamin D analogues on the activity of the hBNP gene promoter. Neonatal rat ventricular myocytes were transfected with -1595 hBNP LUC (0.25 μg) through electroporation as previously described. CMV-Renilla luciferase (0.25 μg) was co-transfected to normalize the samples for differences in transfection efficiency, as described above. In the selected cases, the expression vectors for both the human vitamin D receptor (hVDR) (0.3 μg) and the human retinoid X receptor (hRXR) (0.3 μg) were co-transfected with the BNP-luclferasa report. When the samples were identical, they were treated with endothelin (10 ~ 7 M) or one of the vitamin D analogues. The results are shown in Figures 10 and 11.

Measurement of the activity of Cdk2. Cells were treated with the vehicle or the vitamin analogues D in the indicated intervals. The cells were used with lysis pH regulator and 100 μg of supernatant protein was incubated with 1 μg of anti-Cdk2 antibody and 10 μl of G-Sepharose protein for 1-2 hours at 4 ° C. Immune complex kinase as were performed as previously described using the above-generated immunoprecipitates together with 2 μg of histone 1 and β 32 P-ATP in kinase pH buffer.The reaction products were separated on SDS-denaturing gels polyacrylamide which was then dried and exposed to an X-ray film. The results are shown in Figure 12. This study indicates that VDRAs possess functional activity in the cardiovascular system that is similar, both qualitatively and quantitatively, to that previously demonstrated for a native hormone, 1,25 dihydroxyvitamin D. specifically, the main findings of this study indicate that VDRAs: 1) increases the activity of the type A natriuretic peptide receptor (NPR-A) in neonatal rat aortic smooth muscle cells, 2) increases the promoter activity of the gene NPR-A gene in the same cells through a vitamin D response element, 3) s uprime the ET-dependent stimulation of the BNP gene promoter in cultured neonatal rat ventricular myocytes, 4) inhibit endothelin-dependent stimulation of 3H-thymidine incorporation into DNA and Cdk2 activity in aortic smooth muscle cells adult rat Collectively, these data suggest that paricalcitol, as well as 1,25 dihydroxyvitamin D, may possess cardioprotective effects that control the hypertrophy of cardiac myocytes in the myocardial wall and vascular-protective effects that both limit the proliferation of cells in the vascular wall. of remodeling as they increase the expression / activity of the peptide / NPR anti-proliferative, vasorelaxant-natriuretic system in the vasculature.

Example 5 Changes in Vascular Access in Subjects Treated with Zemplar Methods: A historical group of 2,112 new adult patients for HD, with an AV fistula as the initial primary vascular access, were followed for a period of 35 months (January 1999 to November 2001). ) using a dialysis provider database. The patients were treated with Zemplar or with a therapy without vitamin D; Patients who received Zemplar therapy received at least 10 doses and stayed on the same therapy. Descriptive summary statistics were used to summarize the baseline characteristics and the total number of vascular access changes per year between treatment modalities. In addition, regression models were used to evaluate the association between therapy with Zemplar and without vitamin D, and the total number of vascular access changes per year. Results: The established data contained 577 patients treated with Zemplar and 1535 patients who did not receive any vitamin D therapy. The total number of vascular access changes averages 0.6 changes per year in patients with the Zemplar treatment and 0.9 changes per year in the patients. patients who did not take vitamin D treatment (p = 0.0034). Negative binomial regression was performed to control the baseline co-variants; this revealed that the group without vitamin D was associated with 28% more vascular access changes than patients treated with Zemplar therapy (p = 0.038).

Example 6 Effects of Deficiency 1a.25- (OH) 7Da Genetics in Pressure Blood and Cardiovascular Phenotype in Attacked Mice CYP27B1 M-a-Hydroxylase) 1a, 25- (OH) 2D3 (calcitriol) has been shown to be a negative regulator of the renin-angiotensin system. In this way, it was believed that perhaps mice lacking CYP27B1 (1-alpha-hydroxylase), a key enzyme in the synthesis of the active form of vitamin D3 (1a, 25- (OH) 2D3), could present a Increased blood pressure in relation to wild-type (WT) baits. Attacked mice (KO) and WT were instrumented with telemetry transmitters; Baseline blood pressure and heart rate were continuously recorded and reported as a mean of 24 hours for 7 days. In a subgroup of animals (n = 4 / group), the heart and kidneys were isolated for quantification of mRNA for the vitamin D receptor (VDR), 25-hydroxyvitamin D-24-hydroxylase (CYP24A1), renin, and precursor of natriuretic peptide A (NPPA) through real-time RT-PCR. Through the 7 telemetry registers, mean arterial pressure (MAP) was significantly increased in KO mice (24-hour group means between 112 + 2 and 116 + 4 mmHg, n = 7) in relation to WT litters ( between 106 +2 and 108 ± 2 mmHg, n = 10). In addition, KO mice showed tachycardia throughout the same period (24-hour group means between 601 + _ 6 and 610 + 7 beats / minute) in relation to the WT controls (between 544 ± 6 and 566 +4 beats) /minute). Heart-to-body weight and LV to body weight ratios also tended to rise for KO mice against WT mice reflecting an increased load on the heart muscle. According to the increase in blood pressure in KO mice, the expression of renal renin mRNA was increased (n = 4) in relation to the WT litters while the expression of VDR mRNA was reduced. NPPA modestly elevated in the hearts of KO animals probably reflecting a compensatory effect in response to hypertension and hypertrophy.

The above results demonstrate that the disruption of the CYP27B1 gene product effectively leading to a deficiency of 1 a, 25- (OH) 2D3, produces a sustained rise in blood pressure and heart rate relative to animals filled with 1-alpha- hydroxylase. These results also suggest that increases in MAP can be mediated through the deregulation of the renin-angiotensin system.

Example 7 Differential Effects of Vitamin D Analogs on Vascular Calcification in Uremic Rats Vitamin D receptor activators (VDRAs) were commonly used to manage secondary hyperparathyroidism associated with chronic kidney disease (CKD). Recent clinical data show that VDRAs provide a survival benefit for stage 5 CKD. In patients with CKD, vascular calcification is usually linked to an unfavorable prognosis. In this study, the calcium and phosphorus content in an aorta isolated from uraemic rats was measured. The consumption of 45Ca in cultured aortic rings was also examined. The 5/6 nephrectomized ones were obtained from the Charles River Labs laboratories (Wilmington, MA). Two weeks after the nephrectomy, the rats were put on a diet containing 0.9% phosphorus and 0.6% calcium for 4 weeks, followed by vehicle treatment (5% ethanoi + 95% propylene glycol, 0.4 ml / kg). , paricalcitol or doxercalciferol at 0.67 μg / kg, ip, 3 times / week, for 2 weeks. Twenty-four hours after the last dosing, blood samples were taken through the tail vein under anesthesia with ketamine (50 mg / kg) and the aorta was collected for calcification studies. The combination of 5/6 nephrectomy and a diet high in phosphorus resulted in hypocalcemia (serum calcium: 1.06 vs 1.28 mmol / L in natural animals), hyperphosphatemia (11.6 vs 7.5 mg / dL) and an elevation of PTH in serum (17.3 times), creatinine (2.9 times) and BUN (3.8 times). After the aortas were removed in a sterile form from the rats, the adventitia was carefully removed and each time it was washed three times with the medium. A segment of each aorta was processed for calcium and phosphorus determination. A separate portion of each aorta was hair, cut into 2-3-mm rings and placed in DMEM containing 0.2 μCi / ml 45Ca for 3 days at 37 ° C. Then, the aortic rings were washed, dried, weighed and then dissolved to determine radioactivity. The results show that: (1) there is a linear correlation between the content of calcium and phosphorus and the consumption of 45Ca in the aorta, (2) there is a modest increase (1.6 times) in the content of Ca, but no significant difference in the consumption of 45Ca or phosphorus content in the group treated with paricalcitol when compared to the vehicle, and (3) the consumption of 45Ca was 40 times higher, the Ca content 10 times higher and the phosphorus content 17 times higher in the group treated with doxercalciferol when compared to the vehicle group. Both paricalcitol and doxercalciferol at 0.67 μg / kg raised iCa (-25%) and suppressed PTH (> 90%). These results suggest that doxercalciferol and paricalcitol exhibit different effects on aortic calcification in uraemic rats.

Example 8 Effect of Vitamin D Analogs on Calcification in Human Vascular Smooth Muscle Cells Patients with chronic kidney disease (CKD) experience a high mortality rate of cardiovascular diseases. Vitamin D receptor activators (VDRAs) such as paricalcitol, doxercalciferol and calcitriol were commonly used to manage secondary hyperparathyroidism associated with CKD. Recent clinical data show that VDRAs provide a survival benefit for patients with CKD in Stage 5. In patients with CKD, vascular calcification is usually linked to a non-favorable prognosis. Previous studies have shown that 1,25 (OH) 2D3 (calcitriol) 10"7 to 10" 9 M induced a dose-dependent increase in the calcification of bovine vascular smooth muscle cells (SMC) in vitro (Joño et al, 2000). In this study, the effect of vitamin D analogues on the consumption of 45Ca in a primary culture of human coronary artery smooth muscle cells (CASMC) was examined. Human cultured primary CASMCs were developed at a confluence of >80% and were used within five passages. Cells were treated with drugs for 5 days in the appropriate medium, and then switched back to the base medium (N1: DMEM containing 1.8 mM Ca + 2 and 0.9 mM PO-4) containing 0.2 μCi / ml 45Ca for 24 hours. Then, the cells were washed with PBS three times and the radioactivity was measured through liquid scintillation. The cells cultured in the N1 medium exhibited a minimum absorption of 45Ca (98 ± 14, n = 5). Cells treated with an induction medium (P1-high phosphorus content: DMEM containing 1.8 mM Ca + 2, 3.8 mM PO-4 and 7.5 U / ml alkaline phosphatase) exhibited a dramatic increase in 45Ca uptake. The effect of the induction medium was dose dependent; a mixture of 90% N1 / 10% P1 or 60% N1 / 40% P1 increased the absorption of 45Ca by 7.4 and 34.4 times, respectively. The paricalcitol at 100 nM did not show a significant effect on the uptake of 45Ca in cells treated with the N1 medium or different doses of the P1 medium (20-60%). As a control, paricalcitol stimulated CYP24A1 and suppressed the expression of PA1-1 mRNA in a dose-dependent manner. When the cells are in the N1 or 60% N1 / 40% P1 media, they were treated with high concentrations of activated paricalcitol, calcitriol or doxercalciferol (1-100 nM), with no significant effects on the absorption of 45Ca from the drugs. . These results suggest that human CASMCs grown in medium containing high phosphorus plus alkaline phosphatase, exhibit increased calcium absorption, and vitamin D analogues have no significant effect.

EXAMPLE 9 Differential Effects of Paricalcitol and Doxercalciferol on PTH in Serum and Ionized Calcium in Uraemic Rats with Established Secondary Hyperparathyroidism It is clinically important to suppress elevated levels of PTH in serum in patients with ESRD with established secondary hyperparathyroidism (SHPT) independent of hypercalcemia.

In addition, the model of 5/6 uraemic rats has effectively predicted the clinical profile of vitamin D analogues with respect to PTH and hypercalcemia. In this way, a direct comparison of paricalcitol (PARÍ) and doxercalciferol (DOX) was conducted in male Sprague Dawley rats subjected to 5/6 nephrectomies (surgical ablation) placed on a high phosphorus diet for 4 weeks (0.9% fos ., 0.6% Ca) to establish SHPT. On Day 0, the rats received vehicle (VEH, 5% ethanol, 95% propylene glycol, 0.4 ml / kg, IP) or drug (0.083, 0.17, 0.33, 0.66 mcg / kg, n = 7-10 / group) 3 times / week for 12 days. Blood samples were taken 24 hours after the dose (Day 13). Values of Day 0 (mean ± SEM) for: creatinine (VEH versus false) were 1.02 ± 0.05 vs. 0.48 + 0.01 mg / dL; PTH, 1320 + 185 vs 178 ± 11pg / ml; ionized calcium in blood (iCa, 1.26 + 0.01 vs 1.30 + 0.01 mmol / L); phosphorus (8.70 ± 0.34 vs 7.36 ± 0.09 mg / dL). In VEH, the ratio of iCa values of Day 13 / Day 0 declined to 0.93 + 0.02 vs 1.02 + 0.01 for false; PTH and phosphorus tend to increase. In contrast, the dose-dependent DOX was increased in Ca + + ratios Day 13 / Day 0 to 1.09 + 0.03 * and 1.17 ± 0.04 * to 0.33 and 0.66 mcg / kg dose. PARI did not have any effect on iCa at 0.33 mcg / kg, while the ratio of Ca increased modestly to 1.05 + 0.02 * # at 0.66 mcg / kg, less than that of DOX. The PTH Day 13 / Day 0 ratios for DOX fell to 0.43 ± 0.12, 0.26 + 0.06 *, and 0.22 + 0.11 * in response to 0.18, 0.33, 0.66 mcg / kg, respectively, while PARI increased the PTH ratios to 0.37 ± 0.07 *, 0.33 + 0.06 *. 0.13 + 0.02 *. The level of phosphorus in the serum was not affected in any treatment group. DOX produced a dose-dependent increase in the transport of active calcium in the duodenum ex vivo; PARI had no effect. Thus, in the uraemic rat with SHPT, established, DOX suppressed serum PTH levels according to ionized calcium dose dependent evaluations greater than those caused by equal doses of PARI. These doses, PARÍ effectively reduces PTH without producing dose-dependent changes in calcium in ionized blood. * = p < 0.05 vs. VEH ANOVA; # p < 0.05 odd test DOX vs PARÍ.

Example 10 Inhibition of Renin Biosynthesis Via the Vitamin D Analogue Paricalcitol The renin-angiotensin (RAS) system plays an essential role in the regulation of electrolyte and volume homeostasis. The over-activation of RAS is associated with high blood pressure and other cardiovascular and renal diseases such as cardiac hypertrophy and diabetic nephropathy. Renin is the first limiting rate enzyme of the renin-angiotensin cascade and thus represents an important therapeutic target; however, renin inhibitors are currently not available. It has been shown that 1, 25-dihydroxyvitamin D3 [1, 25 (OH) 2D3], the hormonal form of vitamin D, is an endocrine suppressor of renin biosynthesis, which provides a molecular basis for exploring the potential of vitamin analogues D as renin inhibitors to control RAS.

In the present study, an investigation of the in vitro and in vivo activity of paricalcitol, a lower calcemic vitamin D analog approved by the FDA, to inhibit renin biosynthesis was made. Up to this point, As4.1-hVDR cells, a cell line of JG cell type stably transfected with human VDR, were treated with paricalcitol or 1,25 (OH) 2D3 at doses ranging from 10"10 to 10 ~ 7 M for 24 hours, and its renin inhibitory activity was determined through Northern stain analysis; the cells were also transfected with a renin-luciferase gene promoter reporter plasmid, and the luciferase activity was determined in the presence and absence of paricalcitol or 1,25 (OH) 2D3. These in vitro studies demonstrate that paricalcitol suppresses renin mRNA expression and renin gene transcription in a dose-dependent manner, with a potency comparable to that of 1,25 (OH) 2D3. To determine the in vivo activity of this compound, male CD-1 mice (n = 7) were treated with paricalcitol by intraperitoneal injection and at doses of 1.5 and 3.0 μg / kg of body weight, 3 times a week during 3 weeks, and changes in body weight, ionized calcium in blood, renal renin mRNA and plasma renin activity (PRA) were analyzed at the end of the treatment. These data showed that paricaicitol at these two doses had no effect on body weight and only slightly increased levels of ionized calcium in blood; however, the paricalcitol at these two doses significantly reduced the levels of both renal renin mRNA (from 23% to 45%) and PRA (from 20% to 70%) in the treated mice. These data establish, in principle, that paricalcitol can actually inhibit renin biosynthesis and suggest that paricalcitol can potentially be used to control renin production.

EXAMPLE 11 Effect of Paricalcitol on Expression of Renin mRNA in Vitamin D Deficient Rats Patients with chronic kidney disease (CKD) have a much higher risk of cardiovascular disease than the general public. Several vitamin D receptor activators, including paricalcitol, are currently available for the treatment of hyperparathyroidism secondary to CKD. Recent clinical data show that paricalcitol provides a survival benefit for patients with CKD. Previously, it has been shown that vitamin D receptor activators suppress renin expression in AS4.1 cells. To investigate whether paricalcitol, at non-calcemic doses, can regulate renin in vivo, the expression of renin mRNA in the kidney of vitamin D deficient rats treated with paricalcitol was examined. Male Sprague-Dawley rats housed in an environment free of ultraviolet light were fed with a diet of 0.4% Ca, 0.3% P, deficient in vitamin D, for 5 days, then changed to a diet of 0.02% Ca , 0.5% of P, vitamin D deficient for 21 days. The rats were dosed with 0.003, 0.01, 0.03, and 0.3 μg / kg of paricalcitol, i.p., three times a week for 2 weeks. Only one vehicle (5% ethanol / 95% propylene glycol) was provided to an animal control group, and untreated animals of matching age on a normal diet (0.5% Ca, 0.4% P) served as normal by control. Twenty-four hours after the last dose, the rats were anesthetized with ketamine (100 mg / kg), cardiac bleeding was performed, they were sacrificed through CO2, the tissues were harvested for RNA extraction, and RT-analysis was performed. Real-time PCR, and serum was taken for the determination of calcium, phosphorus and PTH content. Treatment with diet resulted in hlpocalcemla (ionized calcium 0.70 ± 0.02 vs. 1.34 ± 0.01 mmol / L in normal control), elevated PTH in serum (29 times) and phosphorus (9.3 ± 0.3 vs. 8.1 ± 0.2 mg / dl) ), and an increase in renal renin mRNA (+ 20%). Paricalcitol at tested doses did not result in a significant change in calcium, but reduced PTH in the serum depending on dose. Treatment with paricalcitol normalizes the expression of renal renin mRNA at levels found in animals with sufficient normal vitamin D. The above data show that, in rats deficient in vitamin D with elevated PTH and hypocalcemia, paricalcitol at doses that do not affect the serum calcium level, significantly suppress the expression of renal renin mRNA. The effect of paricalcitol on the suppression of renin expression may be a contributing factor to mortality and reduced risk of morbidity in patients with CKD treated with paricalcltol.

Claims (29)

1. A sustained release pharmaceutical composition for preventing, treating and delaying the progression of cardiovascular, cerebrovascular and peripheral vascular diseases, especially heart failure, cardiomyopathy, atherosclerosis, myocardial infarction and cerebrovascular accident, comprising: a therapeutically effective amount of a VDRA or the like of vitamin D; and optionally a therapeutically effective amount of at least one member of the group consisting of an angiotensin-converting enzyme inhibitor, an angiotensin (II) receptor (I) blocker, and an aldosterone blocker.

2. A sustained release pharmaceutical composition according to claim 1, wherein said VDRA or vitamin D analogue is selected from the group consisting of paricalcitol, calcitriol and doxercalciferol.

3. A sustained release pharmaceutical composition according to claim 1, wherein said composition is in the form of a transdermal patch.

4. A sustained release pharmaceutical composition according to claim 1, wherein said composition is in the oral dosage form.

5. A sustained release pharmaceutical composition according to claim 1, wherein said composition is in the subcutaneous dose form.

6. A sustained release pharmaceutical composition according to claim 1, wherein said composition is in an injectable dosage form.

7. A sustained release pharmaceutical composition according to claim 6, wherein said injectable dose form is a member of the group consisting of a subcutaneous dose form and a reservoir dose form.

8. A sustained release pharmaceutical composition according to claim 5, wherein said composition is in the form of an implant.

9. A pharmaceutical composition for the treatment, prevention or delay of the progression of a cardiovascular disease in a mammal, comprising: a therapeutically effective amount of a vitamin D receptor activator or a vitamin D analogue; and optionally a therapeutically effective amount of at least one member of the group consisting of an angiotensin-converting enzyme inhibitor, an angiotensin receptor (II) blocker (I), and an aldosterone blocker.

10. The pharmaceutical composition according to claim 9, wherein said cardiovascular disease is selected from the group consisting of cardiac failure, cardiomyopathy, atherosclerosis, myocardial infarction, cerebrovascular accident and peripheral vascular disease. The pharmaceutical composition according to claim 9, wherein said vitamin D receptor activator or vitamin D analog is selected from the group consisting of paricalcitol, calcitriol and doxercalciferol. 12. The pharmaceutical composition according to claim 9, wherein said composition is in the form of a transdermal patch. 13. The pharmaceutical composition according to claim 9, wherein said composition is in oral dosage form. The pharmaceutical composition according to claim 9, wherein said composition is in a subcutaneous dose form. 15. The pharmaceutical composition according to claim 9, wherein said composition is in an injectable dosage form. 16. The pharmaceutical composition according to claim 15, wherein the injectable dosage form is a member selected from the group consisting of a subcutaneous dose form and a reservoir dose form. 17. The pharmaceutical composition according to claim 14, wherein said composition is in an implant form. 18. A method for preventing, treating and delaying the progression of a vascular disease in a mammal, comprising the step of administering to said mammal a pharmaceutical composition according to claim 9. 19. The method according to claim 18, where the step of administering is continuous. The method according to claim 18, wherein the step of administering is performed using a transdermal patch. The method according to claim 18, wherein the step of administering is carried out using an oral dosage form. 22. The method according to claim 18, wherein the step of administering is carried out using an injectable dose form. The method according to claim 18, wherein the step of administering is carried out using a subcutaneous dose form. 24. A method for treating, inhibiting or preventing vascular disease in a mammal by reducing the expression of PAI-1 in said mammal, comprising the step of administering to said mammal an effective amount of a vitamin D receptor activator or analog of vitamin D. 25. The method according to claim 24, wherein the vitamin D receptor activator is paricalcitol or calcitriol. 26. The method according to claim 24, wherein the vitamin D analog is doxercalciferol or alfacalcidol. 27. A method of treating, inhibiting or preventing thrombosis in a mammal in need of such treatment, inhibition or prevention, comprising the step of administering to said mammal an effective amount of a vitamin D receptor activator or a vitamin analogue. D. 28. The method according to claim 27, wherein the vitamin D receptor activator is paricalcitol or calcitriol. 29. The method according to claim 27, wherein said vitamin D analog is doxercalciferol or alfacalcidol.